. 2023 Nov 20;13(19):e4840. doi: 10.21769/BioProtoc.4840

Table 3. Schwann cells (SC)-specific bioassays.

Table 3 summarizes our suggested assays to reveal the unique responses of cultured hSCs. All assays rely on the detection of one or more inducible molecular readouts while exploiting the experimental fine-tuning of an activating signal (input) driving a measurable change (output) through a SC-specific mechanism of action.

Bioassay	Expected results	Interpretation
Kinase activation (heregulin- dependent)	Cultured hSCs respond to β1-heregulin by rapidly activating ERK and Akt. The differential immunolabeling between control (without heregulin) and treated cells (heregulin-stimulated) should be obvious in all hSCs.	Inter-experimental variability is expected due to the rapid kinetics of ERK and Akt phosphorylation, but the overall responses should be consistent across hSC populations. For a reference, include positive controls to reveal maximal ERK and Akt activation.
Proliferation (heregulin and forskolin-dependent)	β1 heregulin is sufficient to increase the percentage of dividing hSCs. This response is synergistically enhanced by forskolin but forskolin alone does not increase hSC proliferation. hSCs incorporate EdU in a nonsynchronous manner starting roughly at 18–20 h post stimulation under these conditions.	The proportion of proliferating (EdU⁺) cells is donor- and passage-dependent, but it is usually < 40% in early-passage cultures under the suggested conditions. High basal proliferation (in the control condition) may indicate an excess of fibroblasts or, in rare cases, cancerous (heregulin-independent) hSC proliferation.
Differentiation (CPT-cAMP dependent)	CPT-cAMP induces upregulation of certain myelin-associated markers (e.g., O4, Krox20, PRX) and downregulation of immature hSC markers (e.g., cJun) within 3–5 days. Corresponding changes in mRNA expression occur at earlier time points. CPT-cAMP treated hSCs become post-mitotic and morphologically dissimilar to untreated cells.	Select the markers that provide the highest resolution for detection. Assessment of myelin gene expression is only relevant when comparing control (no CPT-cAMP) and CPT-cAMP-treated conditions. The magnitude and kinetics of the expression of different gene products are variable. Substantial batch variability is also expected. Molecular changes may be evident even when morphological differentiation is not apparent.
Senescence (passage-dependent)	hSC populations contain a proportion of senescent cells even at passage-zero. Late-passage cultures may consist only of senescent hSCs. Fibroblasts do not get senescent under these conditions and can be discriminated from hSCs by their negative SA-β-Gal staining.	Normal hSC cultures (nerve-derived) become senescent, usually after four rounds of passaging. The percentage of senescent cells varies from culture to culture or donor to donor, even when same-passage cultures are compared.

Bioassay

Expected results

Interpretation

Kinase activation (heregulin-

dependent)

Cultured hSCs respond to β1-heregulin by rapidly activating ERK and Akt.

The differential immunolabeling between control (without heregulin) and treated cells (heregulin-stimulated) should be obvious in all hSCs.

Inter-experimental variability is expected due to the rapid kinetics of ERK and Akt phosphorylation, but the overall responses should be consistent across hSC populations. For a reference, include positive controls to reveal maximal ERK and Akt activation.

Proliferation (heregulin and forskolin-dependent)

β1 heregulin is sufficient to increase the percentage of dividing hSCs. This response is synergistically enhanced by forskolin but forskolin alone does not increase hSC proliferation.

hSCs incorporate EdU in a nonsynchronous manner starting roughly at 18–20 h post stimulation under these conditions.

The proportion of proliferating (EdU⁺) cells is donor- and passage-dependent, but it is usually < 40% in early-passage cultures under the suggested conditions.

High basal proliferation (in the control condition) may indicate an excess of fibroblasts or, in rare cases, cancerous (heregulin-independent) hSC proliferation.

Differentiation (CPT-cAMP dependent)

CPT-cAMP induces upregulation of certain myelin-associated markers (e.g., O4, Krox20, PRX) and downregulation of immature hSC markers (e.g., cJun) within 3–5 days. Corresponding changes in mRNA expression occur at earlier time points.

CPT-cAMP treated hSCs become post-mitotic and morphologically dissimilar to untreated cells.

Select the markers that provide the highest resolution for detection. Assessment of myelin gene expression is only relevant when comparing control (no CPT-cAMP) and CPT-cAMP-treated conditions. The magnitude and kinetics of the expression of different gene products are variable. Substantial batch variability is also expected.

Molecular changes may be evident even when morphological differentiation is not apparent.

Senescence (passage-dependent)

hSC populations contain a proportion of senescent cells even at passage-zero. Late-passage cultures may consist only of senescent hSCs.

Fibroblasts do not get senescent under these conditions and can be discriminated from hSCs by their negative SA-β-Gal staining.

Normal hSC cultures (nerve-derived) become senescent, usually after four rounds of passaging.

The percentage of senescent cells varies from culture to culture or donor to donor, even when same-passage cultures are compared.